DESCRIPTION (Applicant's abstract): Obesity, diabetes and related diseases are becoming increasingly prevalent in American society. To treat these diseases it is essential that novel signaling molecules involved in the regulation of energy balance be identified and characterized. The recently discovered human agouti-related protein (AGRP) is an orexigenic, paracrine signaling molecule that functions as an endogenous antagonist of melanocortin receptors. AGRP was identified in 1997 by its homology to the more widely studied agouti protein. While both proteins are potent melanocortin receptor antagonists, each selects for a different spectrum of receptor subtypes. Agouti and AGRP both act on melanocortin-4 receptors which are directly linked to body weight control. Unlike the agouti protein, however, AGRP is expressed primarily in the brain. Thus, the interaction between AGRP and the melanocortin-4 receptor is emerging as a focal point for research into the control of energy homeostasis. AGRP's C-terminal domain contains the necessary determinants for antagonism of melanocortin receptors. In 1999, the PI's lab reported the first 3D structure of this domain. This structure reveals a novel fold of three loops emerging from a core rich with disulfide bonds. From analysis of this structure, it is hypothesized that one ioop is essential for melanocortin receptor antagonism and a flanking loop confers AGRP's unique receptor subtype selectivity. This hypothesis will be tested using several complementary experiments. First, the structure of the relevant agouti protein C-terminus will be determined. Next, minimized AGRP's designed to present only the proposed biologically active loops will be prepared and evaluated for structure and activity. Finally, specific residues within the loop that putatively controls antagonist activity will be modified to determine their influence on melanocortin receptor function. The long term objective is to reveal the molecular determinants of AGRP's unique function as an endogenous antagonist and how it selects for specific melanocortin receptors. The findings should play a pivotal role in the understanding and treatment of diseases related to energy balance.